Proc. Nat. Acad. Sci. USAVol. 68, No. 5, pp. 1083-1088, May 1971

Assignments in the Carbon-13 Nuclear Magnetic ResonanceSpectra of Vitamin B12, Coenzyme B12, and Other Corrinoids:Application of Partially-Relaxed Fourier Transform Spectroscopy

DAVID DODDRELL AND ADAM ALLERHAND*

Department of Chemistryt, Indiana University, Bloomington, Ind. 47401

Communicated by John D. Roberts, March 4, 1971

ABSTRACT High-resolution Fourier transform NMRat 15.08 MHz was used to observe the proton-decouplednatural-abundance 13C spectra of aqueous solutions ofcobinamide dicyanide (0.067 M), cyanocobalamin (0.024M), dicyanocobalamin (0.14 M), and coenzyme B12 (0.038M). Assignments were made with the aid of chemical shiftcomparisons, off-resonance single-frequency proton de-coupling, partially-relaxed Fourier transform spectra, andsplittings arising from 13C-31P coupling.As expected, the 13C spectra of the corrinoids were

appreciably more informative than the correspondingproton spectra. Nearly all the lines in the 13C spectra of thecorrinoids were well-resolved single-carbon resonances,in spite of the structural complexity.

Partially relaxed 13C Fourier transform NMR spectra,which yield spin-lattice relaxation times of each resolvedresonance, were found to be a very useful addition to thearsenal ofNMR techniques.

Proton nuclear magnetic resonance (NMR) has been usefulfor investigating the solution properties of vitamin B12(cyanocobalamin, Fig. 1A) and other corrinoids (1-4). How-ever, even at 220 MHz, proton NMR spectra of corrinoidscontain many overlapping lines, as a result of a small range ofchemical shifts and complex spin-spin splittings (4). For thestudy of complex molecules, proton-decoupled spectra of 13Cnuclei in natural abundance are, in general, much moreresolved and simpler to analyze than the corresponding protonspectra (5). The enhanced sensitivity of the Fourier transformtechnique (6) relative to continuous-wave N1\IR has expandedthe range of accessible 13C spectra (7). We will show that byusing the Fourier transform method it is easy to obtain highsignal-to-noise ratios on single-carbon resonances in theproton-decoupled natural-abundance 13C spectra of corrinoids,even when the concentration is limited to 0.02 M.There are only two well-known generally applicable aids to

the assignment of 13C resonances in complex molecules:comparisons within a series of compounds with similar struc-tures, and the use of splitting patterns arising from incompleteproton-decoupling (5). The former is often complicated byuncertainties in chemical shift changes with structure; thelatter is difficult to use when there are many overlapping lines(5). We show below that 13C partially-relaxed Fourier trans-form (PRFT) spectra (8, 9) provide additional help in makingassignments. Intensities in PRFT spectra are given (10) by

A = Ao [1 - 2 exp(- rT1)], (1)where A and Ao are the observed and equilibrium intensities,

Abbreviation: PRFT, partially-relaxed Fourier transform.* Address inquiries to this author.t Contribution No. 1919.

1083

respectively, r is the interval between the 1800 and 900 pulse(8), and T, is the 13C spin-lattice relaxation time. Thus, if twocarbons have different T, values, the intensity of their reso-nances will have a different dependence on r. We have shown(to be published) that 13C T1 values measured from PRFTspectra can be used to distinguish methine, methylene, andnon-protonated carbons on fused ring structures, and to de-tect groups that have internal motion.

In this paper we present assignments in the proton-de-coupled natural-abundance 13C spectra of cyanocobalamin(Fig. 1A), 5'-deoxyadenosylcobalamin (coenzyme B12, Fig.1B), dicyanocobalamin (Fig. 2B), and cobinamide dicyanide(Fig. 2A).

MATERIALS AND METHODSCyanocobalamin was obtained from Nutritional Biochemicals,Cleveland, Ohio. Dicyanocobalamin was prepared by additionof KCN to cyanocobalamin (11). Coenzyme B12 and cobin-amide dicyanide were kindly supplied by Dr. L. Mervyn ofGlaxo Research Ltd., Great Britain. The equipment has been

FIG. 1. Structures of corrinoids. A, cyanocobalamin (vitaminB12). B, 5'-deoxyadenosylcobalamin (coenzyme B12).

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1084 Chemistry: Doddrell and Allerhand

(A) R=H N X CH3

0 N~O.I CH3(B) R: -P-O HO

It 2

HO- H251

FIG. 2. Structures of corrinoids. A, Cobinamide dicyanide. B,Dicyanocobalamin.

described previously (7, 9). When using the 180'-T-90' pulsesequence (10) needed to obtain PRFT spectra (8, 9), we chosethe recycle time between sequences to be at least three timesthe longest T1 value of interest.

RESULTSCompletely proton-decoupled 1'C spectra of cobinamide di-cyanide, cyanocobalamin, and coenzyme B12 are shown in Fig.3. The completely proton-decoupled and the off-resonancesingle-frequency decoupled (5) spectra of dicyanocobalaminare shown in Fig. 4. Cyanocobalamin (0.024 M) and coenzyme

B12 (0.038 M) were studied as nearly saturated aqueous solu-tions at 650C and 560C, respectively. Cobinamide dicyanideand dicyanocobalamin were examined at 560C, in 0.067 M and0.14M aqueous solutions, respectively.

Chemical shifts of the resonances that were assigned are

given in Tables 1-3. As expected (12), some carbons, up tothree bonds removed from phosphorus, exhibited resolvedsplitting arising from 13C-31P scalar coupling. Observedcoupling constants are given in Table 2. Carbons directlybonded to cobalt are expected to show the effect of scalarcoupling to 59Co (100% abundance, spin 7/2). It is likely thatthe quadrupolar contribution to 1/T1 of the cobalt in corri-noids is sufficiently great so that 2TrJccOTjcO <(1. In this case

no resolved splittings of the carbon resonances should be ob-served, but appreciable residual line broadening may stilloccur (13). Only in the spectrum of the relatively concen-

trated dicyanocobalamin did we detect a resonance assignableto the cyano carbons.

Spin-lattice relaxation times of some carbons in dicyano-cobalamin, obtained from intensities in a series of PRFTspectra, are given in Tables 1 and 2. A few representativePRFT spectra are shown in Fig. 5.

ASSIGNMENTSWe have used several of the following five types of evidence inmaking each assignment: (1) Chemical shift comparisons with

TABLE 1. 'IC chemical shifts of the corrin ring carbons

Chemical shifta

Cobin-Assign- amide Coenzyme Cyano- Dicyano- Ticmentb dicyanide B12 cobalamin cobalamin (see)

5,15 588.0(10) 87.3(21) 85.9(18) 87.6(18) >1.0t90.1(11) 88.7(22) 89.3(19) 89.7(19) > 1.0

10 102.0(12) 98.4(23) 98.5(20) 101.7(20) 0.111 109.8(13) 107.5(26) 108.1(22) 109.6(23) >1.0

19 117.7(14) 118.5(29)d 118.3(25) 117.5(24) 0.102 134.1(16) 134.7(35) 134.1(31) 133.8(32) >1.0

(136.3(17) 136.9(36) 136.7(32) 136.1(33) 0.0823,8,13 137.5(18) 138.0(37) 137.5(33) 137.2(34) 0.099

(139.5(19) 139.8(38) 139.3(34) 139.3(35) 0.11(143.7(20) 142.7(39) 142.0(35) 143.5(36) >1.0

7,12,17 146.0(21) 146.2(40) 145.1(36) 146.0(37) >1.0(146.5(22) 146.2(40) 14.5.9(37) 146.5(38) >1.0

18e 162.3(31) f f 162.2(49) -0.2

a In parts per million upfield from carbon disulfide. Estimatedaccuracy ±0.3 ppm. Carbons 4, 6, 9, 11, 14, and 16, whichresonate below 30 ppm, could not be assigned (see text). Numbersin parentheses are peak designations in Figs. 3-6. b Carbonswritten on the same line could not be assigned on a one-to-onebasis. ¢ 13C spin-lattice relaxation time in 0.14 M aqueous di-cyanocobalamin at 560C and pH 9.8. Estimated accuracy ±25%.d This is the most likely assignment, but 119.0 ppm (peak 30)cannot be excluded as a possibility. e Tentative assignment (seetext). f Could not be identified, because of closely spaced reso-nances in this region.

model compounds such as benziminazole (Table 3), nucleotides(12), and porphyrins (D. Doddrell and W. S. Caughey, unpub-lished results). (2) Spectral comparisons within the corrinoids.(3) Off-resonance single-frequency decoupling (5) for dicyano-cobalamin; not attempted on the more dilute samples.(4) Scalar coupling to "P. (5) Partially-relaxed spectra and 13Cspin-lattice relaxation times. We believe this is the first suc-cessful application of 13C PRFT spectra in assignments of res-onances.

It is safe to assume that at 15.08 MHz, '3C relaxation ofprotonated carbons in large molecules is overwhelminglydominated by dipolar interactions with the directly attachedprotons (14) with T1 given by

1/T, = (h/2T)2yc2YH2NrcH 6Tef , (2)where yc and 'Y are the gyromagnetic ratios of '3C and 'H,rH is the CH distance, N is the number of attached hydrogens,and elff is an effective correlation time for rotational reorienta-tion. Eq. 2 is valid when Tefl is much smaller than the inverseof the proton and carbon resonance frequencies, a conditionwhich holds for the solutions studied here, as can be shown bymeans of linewidth measurements (9). In a rigid fused-ringsystem, Teff is about the same for all ring-carbons.4 Thus, T1 ofprotonated ring-carbons is inversely proportional to thenumber of attached hydrogens. Non-protonated carbons havemuch longer relaxation times than protonated ring-carbons.Carbons of methyl groups and other side-chains capable ofundergoing fast internal reorientation may have appreciablyshorter Teff values than ring-carbons. The net result is thatprotonated side-chain carbons may have T, values much

t Evidence for all the statements in the rest of this paragraph isto be published elsewhere.

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FIG. 3. Proton-decoupled natural-abundance '3C NMR spectra of some corrinoids at 15.08 MHz, obtained by the Fourier transformmethod, with 4096 points in the time domain, and 250 ppm sweep widths. Only the range 6-181 ppm upfield from CS2 is shown. Peaksare numbered consecutively from right to left. A, 0.067 M aqueous cobinamide dicyanide at 560C and pH 7.3, using 10,720 scans with arecycle time of 2.72 see (total time 8.1 hr). B, 0.024 M aqueous cyanocobalamin at 650C, using 17,560 scans with a recycle time of 1.36see (total time 6.6 hr). C, 0.038 M coenzyme B12 at 560C and pH 7.5; 14,216 scans with a recycle time of 2.72 see (total time 10.7 hr).

5 56

Itt

23A31

160I1

FIG. 4. Natural-abundance 13C NMR spectra of 0.14 M aqueous dicyanocobalamin at 560C and pH 9.8, obtained at 15.08 MHz bythe Fourier transform method, with 4096 points in the time domain and 250 ppm sweep widths. Only the range 6.4-186.4 ppm upfieldfrom CS2 is shown. A, Completely proton-decoupled spectrum, using a noise-modulated decoupling radio-frequency field, 16,384 scans,and 0.68-sec recycle time (total time 3.1 hr). Peaks are numbered consecutively from right to left. B, Partially proton-decoupled spectrum,using single-frequency off-resonance decoupling, 23,217 scans, and 1.36 sec recycle time (total time 8.8 hr). (The full intensity of peaks5 and 6 is not shown).

I

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TABLE 2. 13C chemical shifts of the a-ribazole and isopropanol-amine moieties

Chemical shifta

Coenzyme Cyano- Dicyano- TicAssignmentb B12 cobalamin cobalamin (sec)

51.4(12)2 or 52.4(13) 51.6(11) 50.1(10) 0.22

9 54.7(14) 56.7(12) 52.2(11) >1.08 59.5(15) 58.4(13) 59.7(13) >1.05,6 561.7(16) 60.5(14) 60.5(14) >1.0

67.0(17) 63.5(15) 60.9(15) >1.04 74.6(19) 76.9(16) 73.8(16) 0.25

7 82.5(20) 82.0(17) 81.6(17) 0.251t 105.0(24) 106.2(21) 107.0(21) 0.19

4' 111.1(27)d 111.2(23)e 109.4(22)f2', 3 120.0(26)f 118.4(25)e ^.-0.2

| 120.3(27)e 120.6(27)h '0.25' 131.9(34) 132.5(30) 131.5(31) 0.18

CHi 123.2(33) 124.3(29) 121.4(29)i 0.24

CH2k 147.6(41)i 147.8(38)f 147.6(39)e ^-0.1

a In parts per million upfield from carbon disulfide. Estimatedaccuracy 0.3 ppm. Numbers in parentheses are peak designa-tions in Figs. 3-6. In the case of doublets arising from coupling to31p, the number in parentheses refers to the downfield peak ofthe doublet. b Carbons written on the same line could not be as-

signed on a one-to-one basis. The methyl carbon of the isopropanol-amine moiety in cobinamide dicyanide resonates at 173.2 ppm

(peak 36). The methyl carbons of the benziminazole moiety in thecobalamins resonate in the range 173-174 ppm (see text). Incoenzyme B12, C-2 of the benziminazole ring and C-8 of the 5'-deoxyadenosyl group could not be assigned on a one-to-one basis.Ti value in 0.14 M aqueous dicyanocobalamin at 56°C andpH 9.8. The methyl carbons of the benziminazole and isopro-panolamine moieties have Ti values of about 0.8 sec. d Doublet,JPc - 8 Hz. e Doublet, JPc - 6 Hz. f Doublet, JPc - 5 Hz.In the range 118-120 ppm. Overlap with 5'-deoxyadenosyl reso-

nances. h Doublet, JPc - 4 Hz. i Methine carbon of isopro-

panolamine moiety. Resonates at 126.6 ppm (peak 15) in cobin-amide dicyanide. i Doublet, JPc - 3 Hz. k Methylene carbonof isopropanolamine moiety. Resonates at 146.5 ppm (peak 22)in cobinamide dicyanide.

longer than those on the ring backbone. The effect of internalmotion on 1/T1 is most pronounced for carbons at, or near thefree end of a side-chain. These principles are quite useful inmaking spectral assignments in complex molecules.

All the above techniques, when taken together, were suffi-cient to make the assignments given in Tables 1-3, which are

justified below. Three types of carbons should resonate below105 ppm: carbonyls, unsaturated carbons on the corrin ring,and the carbons of the benziminazole ring. The benziminazolering carbons were identified by comparison of the spectra ofcobinamide dicyanide and the cobalamins. PRFT spectrawere used to identify all non-protonated carbons in this region.In the case of dicyanocobalamin, off-resonance single-fre-quency decoupling (Fig. 4) confirmed the identifications of non-protonated and methine carbons. Specifically, C-10 was as-

signed to the resonance at about 100 ppm, because it is theonly protonated unsaturated carbon on the corrin ring, and be-cause its chemical shift is comparable to that of similar car-

bons in porphyrins (D. Doddrell and W. S. Caughey, unpub-lished results). Carbons 5 and 15 of the corrin ring resonateabout 10-15 ppm downfield from C-10, as a result of methyl

TABLE 3. Some 1'C chemical shiftsa

5'-Deoxyadenosylgroup of Adenosine

coenzyme B12 Benziminazoleb 3'-monophosphatec

Assign- Chemical Assign- Chemical Assign- Chemicalmentde shift ment shift mentd shift

6 37.3(9) 2 53.4 1' 104.22 39.9(10) 8,9 57.5 4' 107.6f4 44.1(11) 5,6 62.2 2',3' 5118.5f8' 51.4(12) 4,7 78.9 119.0h

or 52.4(13) CH3 174.6 5' 130.85 74.0(18)

5106.7(25)1 ',4' t 107.5(26)i

a In parts per million upfield from carbon disulfide. Estimatedaccuracy ±40.3 ppm. b Saturated methanol solution, at about420C. Carbon numbering system as in Fig. 1. c 1 M aqueoussolution, pH 13.3, at about 421C. d Carbons written on the sameline could not be assigned on a one-to-one basis. Numbers inparentheses are peak designations in Fig. 3C. e The resonanceof C-5' was not observed, probably because of broadening frominteraction with 59Co (see text). The resonances of C-2' and C-3'overlapped with those of the ribose moiety at 118-120 ppm.f Doublet, Jcp 5 Hz' See assignment of C-2 of the benzimina-zole moiety in Table 2. h Doublet ,Jcp - 4 Hz. i Coincides withC-1 of the corrin ring (see text).

substitution (15). The remaining unsaturated carbons of thecorrin ring are strongly deshielded as a result of direct bondingto nitrogen, and they resonate in a range which overlaps par-tially with the amide carbonyl region (16). Position 2 of thebenziminazole ring was assigned to the resonance at about 51ppm. The remaining six benziminazole ring carbons can bedivided into three pairs. The assignments for C-2 and the threepairs were made from PRFT spectra, and on the basis of chemi-cal shifts in benziminazole itself (Table 3). Differentiationwithin the pairs was done for C-4,7 and C-8,9 by comparisonof the spectra of dicyanocobalamin and cyanocobalamin, on theassumption that the resonances that shift appreciably uponcoordination are closer to the coordinating nitrogen of thebenziminazole ring than resonances that are not shifted. Thebase carbons of the 5'-deoxyadenosyl group in coenzyme B12(Table 3) were assigned (17) by comparison of the spectra ofcyanocobalamin, coenzyme B12, and adenosine. The methylenecarbon directly attached to cobalt in coenzyme B12 was not de-tected, probably as a result of line broadening arising frominteraction with 59Co (see above). The weak but reproduciblebroad resonance centered at about 55.5 ppm in the spectrumof dicyanocobalamin (peak 12 in Fig. 4A) was attributed to theCN carbons.The five saturated non-protonated carbons of the corrin ring

in all the corrinoids were identified from PRFT spectra. Fig. 6illustrates the intensity changes in the resonances of 0.024 Maqueous cyanocobalamin when one goes from the normalspectrum (Fig. 6A) to a PRFT spectrum with T = 1.02 sec(Fig. 6B). At this r value, the resonances of all the non-proto-nated carbons are nulled (T Tiln2), while the relaxation timesof all the protonated carbons are sufficiently short to yield posi-tive signals. Carbon 1 was assigned to the furthest downfieldresonance in this group, because of its direct attachment tonitrogen. Of the remaining four, carbons 7, 12, and 17 are

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*0.68

FIG. 5. Proton-decoupled natural-abundance "C Fourier transform NMR spectra of 0.14 M aqueous dicyanocobalamin (560C, pH 9.8)obtained at 15.08 MHz using 4096 points in the time domain, 250 ppm sweep widths, 2.72 see recycle times, and 4096 scans (3.1 hr) perspectrum. Only the range 6-181 ppm upfield from CS2 is shown in each case. The top spectrum is the normal one, with the same peaknumbering system as in Fig. 4A. The others are PRFT spectra, with r values given in seconds. (For T = 0.17 and 0.04, the full depthbetween peaks 5 and 6 is not shown).

structurally similar and were thus assigned to the threenon-protonated resonances grouped upfield at about 142-147ppm (Table 1). Carbon 2 should be deshielded because of asteric interaction (18) with the methyl group attached to C-1.It was assigned to the resonance at about 134 ppm.

The ribose resonances were identified by comparing thespectra of cobinamide dicyanide and the cobalamins (Fig. 1),and by the presence of resolved splittings caused by 3"P-"3Ccoupling. Specific assignments were made by comparison withthe spectra of adenosine 3'-monophosphate (Table 3), which

FIG. 6. Upfield portion in the proton-decoupled natural-abundance 13C Fourier transform NMR spectra of 0.024 M cyanocobalaminat 61 IC, obtained at 15.08 MHz using 4096 points in the time domain, 125 ppm sweep widths, 2.72 see recycle times, and 16,384 scans(12.4 hr) per spectrum. Only the range 71.2-181.2 ppm upfield from CS2 is shown. To avoid reflection of the downfield resonances notcovered in the 125 ppm sweep width, we used a four-pole sharp cutoff filter, and set the carrier frequency upfield. A, Normal spectrum,with the non-protonated carbons indicated by the peak numbers of Fig. 3B. The resolution is better than in Fig. 3B because of the nar-rower sweep width. (The full intensity of peak 56 is not shown.) B, PRFT spectrum with r = 1.02 sec. The resonances of the non-pro-tonated carbons are nulled, while all the resonances of the protonated carbons are already quite positive.

'IC NMR of Corrinoids 1087

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1088 Chemistry: Doddrell and Allerhand

was chosen because its sugar ring is a reasonable model for thatof a-ribazole. The latter compound was unavailable. In thecase of coenzyme B12, carbons 1', 4', and 5' of the a-ribazolemoiety were specifically assigned (Table 2), but carbons 2'and 3' were not, because they overlapped with the correspond-ing carbons of the 5'-deoxyadenosyl group (Table 3). Carbons1' and 4' of this group were assigned to the resonances at 106.7and 107.5 ppm, but not on a one-to-one basis. The PRFTbehavior of the resonance at 107.5 ppm in coenzyme B12 wasindicative of the coincidence of a protonated and a non-proto-nated carbon. The latter was identified as C-1 of the corrin ring,as discussed above. After the above assignments were complete,the methine and methylene carbons of the isopropanolaminemoiety in the cobalamins were tentatively assigned to theresonances which exhibited what appeared to be couplingto 31p. These assignments were then confirmed by consider-ation of the spectrum of cobinamide dicyanide, which still con-tained these resonances, but as singlets, and with the methinesignal being slightly shifted downfield (Table 2).At this point, the only unidentified resonance below 125

ppm was one at about 118 ppm in all the corrinoids. Its relaxa-tion time in dicyanocobalamin was 0.1 sec, a value com-parable to that of C-10 of the corrin ring (Table 1). The chem-ical shift and short relaxation time were compatible only withan assignment to C-19. It should be noted that off-resonancesingle-frequency decoupling could not be used to identify C-19as a methine carbon, because of the close proximity of otherresonances (Fig. 4). The three resonances at 136-140 ppm wereassigned to methine carbons on the corrin ring on the basis oftheir T, values and off-resonance single-frequency decoupling.These resonances were assigned to the structurally similarcarbons 3, 8, and 13, but not on a one-to-one basis. At thispoint, all resonances below 147 ppm had been identified. Theremaining unassigned carbons were all the methyl carbons, themethylene carbons of the corrin ring side-chains, and only onemethine carbon, namely C-18. In the off-resonance proton-decoupled spectrum of dicyanocobalamin (Fig. 4B), theresonance at about 162 ppm appeared to be a doublet, with thedownfield component partially buried under other resonances.On this basis, it was assigned to C-18. This assignment is onlytentative, because we could not rule out the possibility thatthe apparent doublet was actually a methyl quartet, with theouter lines too weak to be observed. In addition, the T1 valueof this resonance was about twice that of all the other methinecarbons on the corrin ring (Table 1).The two resonances at about 149-151 ppm and the one at

about 158 ppm were assigned to methylene carbons (see Fig.4). It is likely that the downfield resonances correspond tomethylene groups that are directly attached to the corrin ring.On the basis of their chemical shifts, they were tentativelyassigned to the structurally similar methylene carbons at-tached at C-2 and C-7. The carbon resonating at about 158ppm probably belongs to one of the ,-methylene groups atC-3, C-8, or C-13 of the corrin ring. The only other well-resolved methylene carbons in the spectrum of dicyano-cobalamin resonated at 166.1, 167.0, and 167.8 ppm. Theywere tentatively assigned to the three structurally simi-

lar methylenes directly attached to C-3, C-8, and C-13 of thecorrin ring. No further methylene assignments could be made.

If the resonance at about 162 ppm is indeed C-18 (seeabove), then the line at about 154 ppm must arise from amethyl carbon (peak 43 in Fig. 4A). Its chemical shift is con-sistent with that expected for the methyl group at C-1. A com-parison of the spectra of cobinamide dicyanide and the cobala-mins proves that the methyl groups attached to the benzimina-zole ring must be located in the relatively unresolved region at173-174 ppm (peaks 52 and 53 in the spectrum of cyano-cobalamin, Fig. 3B). These chemical shifts are in agreementwith those in benziminazole itself (Table 3), and the T,values of these resonances in dicyanocobalamin (Table 2)are indicative of methyl groups undergoing fast internalreorientation. All spectral lines above 170 ppm were easilyidentified as methyl resonances, but no further specific assign-ments were attempted.

Completion of the assignments should be feasible if addi-tional derivatives are studied.

We are grateful to Dr. L. Mervyn of Glaxo Research Ltd.,Great Britain, for making available some of the corrinoids usedin this study. This research was supported by the NationalScience Foundation (grant No. GP-17966), and by the donorsof the Petroleum Research Fund administered by the AmericanChemical Society (grant No. 4559-ACa).

1. Hill, H. A. O., J. M. Pratt, and R. J. P. Williams, J. Chem.Soc., 1965, 2859.

2. Hill, H. A. O., B. E. Mann, J. M. Pratt, and R. J. P.Williams, J. Chem. Soc. (A), 1968, 564.

3. Penley, M. W., D. G. Brown, and J. M. Wood, Biochemistry,9, 4302 (1970).

4. Cockle, S. A., H. A. 0. Hill, R. J. P. Williams, B. E. Mann,and J. M. Pratt, Biochim. Biophys. Acta, 215, 415 (1970).

5. Reich, H. J., M. Jautelat, M. T. Messe, F. J. Weigert, andJ. D. Roberts, J. Amer. Chem. Soc., 91, 7445 (1969).

6. Ernst, R. R., and W. A. Anderson, Rev. Sci. Instrum., 37, 93(1966).

7. Allerhand, A., D. W. Cochran, and D. Doddrell, Proc. Nat.Acad. Sci. USA, 67, 1093 (1970).

8. Vold, R. L., J. S. Waugh, M. P. Klein, and D. E. Phelps,J. Chem. Phys., 48, 3831 (1968).

9. Allerhand, A., D. Doddrell, V. Glushko, D. W. Cochran,E. Wenkert, P. J. Lawson, and F. R. N. Gurd, J. Amer.Chem. Soc., 93, 544 (1971).

10. Abragam, A., The Principles of Nuclear Magnetism (OxfordUniversity Press, Oxford, 1967), p. 64.

11. Hayward, G. C., H. A. 0. Hill, J. M. Pratt, N. J. Vanston,and R. J. P. Williams, J. Chem. Soc., 1965, 6485.

12. Dorman, D. E., and J. D. Roberts, Proc. Nat. Acad. Sci.USA, 65, 19 (1970).

13. Chap. XI, section III-A of ref. 10.14. Kuhlmann, K. F., D. M. Grant, and R. K. Harris, J. Chem.

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16. Horsley, W., H. Sternlicht, and J. S. Cohen, J. Amer. Chem.Soc., 92, 680 (1970).

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18. Dalling, D. K., and D. M. Grant, J. Amer. Chem. Soc., 89,6612 (1967).

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